Our revised project High Throughput Bioengineering of Detoxification Enzymes focuses on organophosphate hydrolases, which have immediate value for mediation or detection of chemical warfare agents (CW; i.e., nerve gases), and long term value for dealing with contaminating pesticides in humans and the environment. Two candidate organophosphate hydrolases have been structurally determined; a TIM barrel-like, dimeric organophosphate hydrolase (OPH) from Pseudomonas diminuta, and a 13-propeller peptide diisopropylfluoro-phosphatase (DFPase) from Lo/igo vu/garis (squid). Each has some proven, but inefficient, effect against organophosphate CW agents and pesticides, and modification of certain residues has been shown to increase their ability to hydrolyze some of these agents [1, 3, 3b, 11-15]. We propose to use our patented technologies for seamless gene assembly (TOPPs) to generate a large library of designed, substrate-specific substitutions (DPSSs). By making multiple modifications in 24-25 residues shown lining the active sites of both OPH and DFPase [1,2,3], we will generate at least 1,000 modified genes for each in 6 months. By screening the expressed genes for their enzyme kinetics with 3-4 substrates (including pesticides and surrogate CW agents), data will be available for designing new modifications in these sets. Preliminary data have already been integrated into our iterative, primer design, and we are preparing to automate the whole process. Our Phase I work will be to expand enormously the number of mutants available for both enzymes, and we believe this approach is a novel way to produce the desired improvements. Phase II aims will likely include i) verification of enzyme activities in the resource library, ii) further analyses of their stability, optimal conditions of assay and other substrates (by us or by collaborators), and iii) finally adding adaptors and modifications to these enzymes for use as biosensors or as a detoxification and/or decontamination tools.